Polystyrene-coated micropallets for culture and separation of primary muscle cells
Polystyrene-coated micropallets for culture and separation of primary muscle cells
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DOI：
10.1007/s00216-011-5596-9
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发表时间：
2012-01-01
影响因子：
4.3
通讯作者：
Allbritton, Nancy L.
中科院分区：
文献类型：
--
作者：
Detwiler, David A.;Dobes, Nicholas C.;Allbritton, Nancy L.
尽管鉴定了大量的成体干细胞类型，但目前的原代细胞分离和鉴定技术产生异质性样品，使得详细的生物学研究具有挑战性。为了鉴定分离细胞的亚群，需要能够同时进行细胞培养和克隆的技术。Micropallet阵列是一种新型的贴壁细胞克隆平台，具有巨大的潜力。然而，组成这些阵列的微结构由环氧光致抗蚀剂1002 F制造，环氧光致抗蚀剂1002 F是不适合于许多细胞类型的生长表面。对微结构的表面性质进行优化，以培养卫星细胞，即需要改进细胞分离技术的原代肌细胞。各种表面材料进行了筛选卫星细胞的粘附和增殖，并比较其最佳基板，明胶涂层的培养皿。通过接触印刷将1 μ m厚的聚苯乙烯共聚物施加到微结构上。发现在95%苯乙烯中的5%丙烯酸的带负电荷的共聚物与用于细胞粘附和增殖的对照皮氏培养皿相当。在对照培养皿和最佳共聚物涂覆的表面上培养的细胞保持未分化状态，并且在标准分化方案期间显示指示细胞分化的两个基因的类似mRNA表达。使用额外的细胞外基质蛋白质胶原和明胶的接触打印层的实验显示没有进一步的改善。这种微托盘涂覆策略易于适用于优化其他类型原代细胞的阵列表面。
Despite identification of a large number of adult stem cell types, current primary cell isolation and identification techniques yield heterogeneous samples, making detailed biological studies challenging. To identify subsets of isolated cells, technologies capable of simultaneous cell culture and cloning are necessary. Micropallet arrays, a new cloning platform for adherent cell types, hold great potential. However, the microstructures composing these arrays are fabricated from an epoxy photoresist 1002F, a growth surface unsuitable for many cell types. Optimization of the microstructures' surface properties was conducted for the culture of satellite cells, primary muscle cells for which improved cell isolation techniques are desired. A variety of surface materials were screened for satellite cell adhesion and proliferation and compared to their optimal substrate, gelatin-coated Petri dishes. A 1-mu m thick, polystyrene copolymer was applied to the microstructures by contact printing. A negatively charged copolymer of 5% acrylic acid in 95% styrene was found to be equivalent to the control Petri dishes for cell adhesion and proliferation. Cells cultured on control dishes and optimal copolymer-coated surfaces maintained an undifferentiated state and showed similar mRNA expression for two genes indicative of cell differentiation during a standard differentiation protocol. Experiments using additional contact-printed layers of extracellular matrix proteins collagen and gelatin showed no further improvements. This micropallet coating strategy is readily adaptable to optimize the array surface for other types of primary cells.